Antimicrobial peptides (AMPs) are present in all species investigated to date. They form an important part of innate immunity, protecting the organism from infection by directly killing invading bacteria. Since pathogenic microorganism show an increasing tendency to be immune against common antibiotics, AMPs carry remarkable pharmaceutical promise as next-generation antibiotics.
The electron micrographs below show how a bacterium is affected by LL-37, a human antimicrobial peptide. The bacterium dies if a threshold called 'minimum inhibitory concentration' (MIC) is reached. Even at concentrations below the MIC, the bacterium shows visible damage.
The exact mechanism by which AMPs kill microorganisms is still under debate. The cartoon below summarizes aspects known for the action of AMPs. In solution (top row of the cartoon), AMPs may be found in unordered conformation as well as in defined secondary structure. In some cases, AMPs aggregate to form oligomers in solution.
Binding to microbial membranes is the first step for AMP to kill bacteria. Currently it is not known which conformation in solution leads to membrane binding. After binding, all AMP display defined secondary structure. Regularly, membrane-bound AMP are observed to form dimers or higher oligomers.
After binding, AMP make bacterial membranes permeable for ions and other cellular content, thus harming or killing the bacterium. Two molecular models to explain the membrane permeability caused by AMP are illustrated on the bottom of the cartoon. AMPs may act as detergent, seriously perturbing the structural integrity of the membrane ('carpet model', left). Another possible way of action may be the formation of actual protein-lined membranes pores ('barrel stave model', right). Our lab has studied a large variety of antimicrobial peptides with all sorts of secondary structure. Currently, we are focusing on two human AMPs, HBD and LL-37.
Human AMPs: Dozens of AMPs are found in the human organism. The alpha- and beta-defensins form the largest group. The cathelicidin group, found in all species investigated, has only a single representative in human, called LL-37. Both LL-37 and beta-defensins are under investigation in our lab.
Human beta-defensin 3 (HBD-3) is an antimicrobial peptide with a complex molecular structure, shown left in the figure below. The structure consists of a three-stranded b-sheet, an a-helical portion and several disordered loops. Also shown are the three disulfide bridges that stabilize the molecular structure. HBD-3 is known to form dimers in solution, a model is shown on the right of the figure. Currently we investigate several mutants and fragments of HBD-3 in order to identify minimal structural motifs needed to be active against bacteria.
The human cathelicidin LL-37 is an alpha-helical amphipathic peptide. The figure below shows an electrostatic surface plot and a cartoon representation of LL-37, stressing the amphipathic nature of the peptide. The observed topology with a hydrophilic surface on the top and a hydrophobic surface on the bottom of the peptide is a common feature of all antimicrobial peptides. It allows for membrane binding with the hydrophobic face of the AMP inserting into the hydrophobic core of the lipid bilayer. Note that this amphipathic topology is disturbed at the N-terminus of LL-37. This altered topology may be necessary for LL-37 to perform the complex signaling function which it serves in addition to its antimicrobial functions.
We have investigated LL-37's structure in lipid bilayers using 13C- and 15N-labeled LL-37. The 15N-NMR spectra of aligned and unaligned samples (bottom left) show that LL-37 aligns with the amphipathic bilayer interface. In addition, 2H- and 31P-NMR of the bilayer lipids revealed a severe distortion of the hydrophobic bilayer core in the presence of LL-37. The cartoon on the right summarizes the findings of bilayer perturbation and surface alignment of the peptide.
Currently we hope to extend these studies by the use of uniformly 15N-labeled LL-37, and by experiments designed to detect and characterize a probable oligomerization of LL-37.
Publications on this project
- Porcelli F, Ramamoorthy A, Barany G and Veglia G. On the role of NMR spectroscopy for characterization of Antimicrobial peptides, Membrane Proteins:Methods in Molecular Biology. 1063(2013) 159-180.
- Ramamoorthy A, Jiadi X. 2D 1H/1H RFDR and NOESY NMR Experiments on a Membrane-Bound Antimicrobial Peptide Under Magic Angle Spinning, J. Phys. Chem. B. 117(2013) 6693-6700.
- Ding B, Soblosky L, Nguyen K, Geng J, Yu X, Ramamoorthy A, Chen Z. Physiologically-Relevant Modes of Membrane Interactions by the Human Antimicrobial Peptide, LL-37, Revealed by SFG Experiments. Scientific Reports. (E-pub).
- Lee DK, Brender JR, Sciacca MF, Krishnamoorthy J, Yu C, Ramamoorthy A. Lipid composition dependent membrane fragmentation and pore-forming mechanisms of membrane disruption by pexiganan (MSI-78). Biochemistry. 52(2013) 3254-3263.
- Epand RF, Maloy L, Ramamoorthy A, Epand RM. Amphipathic Helical Cationic Antimicrobial Peptides Promote Rapid Formation of Crystalline States in the Presence of Phosphatidylglycerol: Lipid Clustering in Anionic Membranes. Biophys. J. 98 (2010) 2564-2573.
- Epand RF, Maloy WL, Ramamoorthy A, Epand RM. Probing the "Charge Cluster Mechanism" in Amphipathic Helical Cationic Antimicrobial Peptides. Biochemistry 49 (2010) 4076-4084.
- Bhunia A, Domadia PN, Hallock KJ, Ramamoorthy A, Bhattacharjya S. NMR Structure of Pardaxin, a Pore-Forming Antimicrobial Peptide, in Lipopolysaccharide Micelles: Mechanism of Outer Membrane Permeabilization. J. Biol. Chem. 285 (2010) 3883-3895.
- Ramamoorthy A, Lee DK, Narasimhaswamy T, Nanga RPR. Cholesterol reduces pardaxin's dynamics-a barrel-stave mechanism of membrane disruption investigated by solid-state NMR. Biochim. Biophys. Acta 2 (2010) 223-227.
- Thennarasu S, Tan AM, Penumatchu R, Shelburne CE, Heyl DL, Ramamoorthy A. Antimicrobial and Membrane Disrupting Activities of a Peptide Derived from the Human Cathelicidin Antimicrobial Peptide LL37. Biophys. J. 98 (2010) 248-257.
- Bhattacharjya S, Ramamoorthy A. Multifunctional host defense peptides: functional and mechanistic insights from NMR structures of potent antimicrobial peptides. FEBS J. 276 (2009) 6465-6473.
- Gottler L and Ramamoorthy A. Membrane Orientation, Mechanism, and Function of Pexiganan - A Highly Potent Antimicrobial Peptide Designed From Magainin. Biochim. Biophys. Acta 1788 (2009) 1680-1686.
- Ramamoorthy A. Beyond NMR spectra of antimicrobial peptides: Dynamical images at atomic resolution and functional insights. Solid State Nucl. Magn. Res. 35 (2009) 201-207.
- Bhunia A, Ramamoorthy A, Bhattacharjya S. Helical Hairpin Structure of a Potent Antimicrobial Peptide MSI-594 in Lipopolysaccharide Micelles by NMR Spectroscopy. Chemistry - A European Journal 15 (2009) 2036-2040.
- Gottler LM, de la Salud Bea R, Shelburne CE, Ramamoorthy A, Marsh EN. Using Flourous Amino Acids to Probe the Effects of Changing Hydrophobicity on the Physical and Biological Properties of the Beta-Hairpin Antimicrobial Peptide Protegrin-1. Biochemistry. 47 (2008) 9243-9250.
- Porcelli F, Verardi R, Shi L, Henzler-Wildman KA, Ramamoorthy A, Veglia G. NMR Structure of the Cathelicidin-Derived Human Antimicrobial Peptide LL-37 in Dodecylphosphocholine Micelles. Biochemistry. 47 (2008) 5565-5572.
- Gottler LM, Lee HY, Shelburne CE, Ramamoorthy A, Marsh ENG. Using Fluorous Amino Acids to Modulate the Biological Activity of an Antimicrobial Peptide. ChemBioChem 9 (2008) 370-373.
- Hoskin DW, Ramamoorthy A. Studies on anticancer activities of antimicrobial peptides. Biochim. Biophys. Acta 1778 (2008) 357-375.
- Shelburne C, An F, Dhople V, Ramamoorthy A, Lopatin D, Lantz M, The spectrum of anti-microbial activity of the bacteriocin Subtilosin A, J. Antimicrob. Chemother., 59 (2007) 297-300.
- Special issue: A. Ramamoorthy (Ed.), Membrane biophysics of antimicrobial peptides, Biochim. Biophys. Acta 1758 (2006) 1177-1540.
- Moon JY, Henzler-Wildman KA, Ramamoorthy A, Expression and purification of a recombinant LL-37 from Escherichia coli, Biochim. Biophys. Acta 1758 (2006) 1351-1358.
- Durr UHN, Sudheendra US, Ramamoorthy A, LL-37, the only human member of the cathelicidin family of antimicrobial peptides, Biochim. Biophys. Acta 1758 (2006) 1408-1425.
- Dhople V, Krukemeyer A, Ramamoorthy A, The human beta-defensin-3, an antibacterial peptide with multiple biological functions, Biochim. Biophys. Acta 1758 (2006) 1499-1512.
- Ramamoorthy A, Thennarasu S, Lee DK, Tan A, Maloy L, Solid-State NMR investigation of the membrane-disrupting mechanism of antimicrobial peptides MSI-78 and MSI-594 derived from Magainin 2 and Melittin, Biophys. J . 91 (2006) 206-216.
- Ramamoorthy A, Thennarasu S, Tan A, Gottipati K, Sreekumar S, Heyl DL, An FYP, Shelburne CE, Deletion of all cysteines in Tachyplesin I abolishes hemolytic activity and retains antimicrobial activity and LPS selective binding, Biochemistry 45 (2006) 6529-6540.
- Porcelli F, Buck-Koehntop B, Thennarasu S, Ramamoorthy A, Veglia G, Structures of the dimeric and monomeric variants of magainin antimicrobial peptides (MSI-78 and MSI-594) in micelles and bilayers by NMR spectroscopy, Biochemistry 45 (2006) 5793-5799.
- Ramamoorthy A, Thennarasu S, Tan A, Lee DK, Clayberger C, Krensky AM, Cell selectivity correlates with membrane-specific interactions: A case study on the antimicrobial peptide G15 derived from granulysin, Biochim. Biophys. Acta 1758 (2006) 154-163.
- Epand RF, Ramamoorthy A, Epand RM, Membrane lipid composition and the interaction of pardaxin: The role of cholesterol, Prot. Pept. Lett. 13 (2006) 1-5.
- Powers JPS, Tan A, Ramamoorthy A, Hancock REW, Solution structure and interaction of the antimicrobial polyphemusins with lipid membranes, Biochemistry 44 (2005) 15504-15513.
- Thennarasu S, Lee DK, Poon A, Kawulka KE, Vederas JC, Ramamoorthy A, Membrane permeabilization, orientation, and antimicrobial mechanism of subtilosin A, Chem. Phys. Lipids 137 (2005) 38-51.
- Thennarasu S, Lee DK, Tan A, Kari UP, Ramamoorthy A, Antimicrobial activity and membrane selective interactions of a synthetic lipopeptide MSI-843, Biochim. Biophys. Acta 1711 (2005) 49-58.
- Porcelli F, Buck B, Lee DK, Hallock KJ, Ramamoorthy A, Veglia G, Structure and orientation of pardaxin determined by NMR experiments in model membranes, J. Biol. Chem. 279 (2004) 45815-45823.
- Henzler-Wildman KA, Martinez GV, Brown MF, Ramamoorthy A, Perturbation of the hydrophobic core of lipid bilayers by the human antimicrobial peptide LL-37, Biochemistry 43 (2004) 8459-8469.
- Wildman KAH, Lee DK, Ramamoorthy A, Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37, Biochemistry 42 (2003) 6545-6558.
- Hallock KJ, Lee DK, Ramamoorthy A, MSI-78, an analogue of the magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain, Biophys. J. 84 (2003) 3052-3060.
- Hallock KJ, Lee DK, Omnaas J, Mosberg HI, Ramamoorthy A, Membrane composition determines pardaxin's mechanism of lipid bilayer disruption, Biophys. J. 83 (2002) 1004-1013.